Objective To prepare human anti-Mullerian hormone (AMH) immunomagnetic beads and HRP-labeled antibodies and establish a rapid double-antibody sandwich ELISA based on nanometer magnetic beads. Methods The expression vector of human AMH protein was constructed, and the recombinant AMH protein was expressed and purified. BALB/c mice were immunized with the recombinant protein to prepare the polyclonal antibody. Spleen cells were fused with myeloma Sp2/0 cells by PEG. Hybridoma cell lines which could stably secret monoclonal antibodies against AMH were screened out by ELISA. Monoclonal antibodies were produced from the ascites fluid of mice injected intraperitoneally with hybridoma cells and evaluated by Western blotting. Polyclonal antibodies purified from protein A were coupled to nano-magnetic' beads and used as capture antibodies, while HRP-labeled monoclonal antibody was prepared by sodium periodate method and used as probe antibody. A double antibody sandwich ELISA based on the nano-magnetic beads was established and optimized. Results A monoclonal antibody with good specificity for AMH was obtained,' and its subtype was IgG2b. The titers of purified polyclonal antibodies and monoclonal antibodies were up to 1:51 200. The capture antibody coupled with magnetic beads and the probe antibody labeled with HRP kept their good activity. The established method could detect AMH antigen within 1 hour and the detection limit was 50 ng/mL. Conclusion The prepared AMH immunomagnetic beads can be used for the fast and visualized detection of recombinant AMH.